Interpretive Summary: Virus-induced gene silencing is a powerful and rapid technique for analysis of gene function in plant growth and development that has been applied to a range of plant taxa. We attempted to use VIGS for functional analysis of genes associated with flower senescence and abscission in Mirabilis jalapa. M. jalapa plants infected with the Tobacco Rattle Virus (TRV) VIGS vector containing a fragment of the M. jalapa phytoene desaturase (PDS) gene did not show the expected photobleaching phenotype. We hypothesized that the failure of VIGS in this species might be due to the presence of the Mirabilis Antiviral Protein (MAP), which is known to have potent activity in preventing viral infection and inhibiting protein synthesis. M. jalapa plants inoculated with a TRV vector containing fragments of PDS and MAP (to silence the antiviral protein) resulted in the typical photo-bleaching phenotype in photosynthetic tissues. We demonstrated reduced transcript abundance (and the expected phenotype) when we used VIGS with the TRV/MAP vector to silence a range of genes. Silencing 3,4-dihydroxy-L-phenylalanine 5-glucosyl transferase (DOPA 5GT) reduced pigmentation in stems, leaves, and flowers. This is the first direct demonstration of the role of enzymes that have been proposed to be key steps in the synthesis of betalain pigments. Silencing endogenous antiviral proteins may open a wider range of taxa to the use of VIGS for gene function analysis.

Technical Abstract:
Virus-induced gene silencing (VIGS) is an attractive and rapid technique for loss of function assay that can reveal the phenotype of embryo-lethal sequences and avoids the need for time consuming transformation and regeneration processes. Among various VIGS vectors that have been explored, the tobacco rattle virus (TRV) vectors has proved to be very useful in a range of plant taxa, including Nicotiana benthamiana, tomato and petunia, Eschscholzia californica, Aquilegia, and Jatropha. The properties of the TRV VIGS vector suggest that it could be utilized in studies of the function of candidate genes in many organs, physiological stages, and plants. But, successful application of VIGS has been limited to a relatively small group of taxa.
We are studying leaf/flower senescence and abscission using Four O’clock (Mirabilis jalapa) as a model plant since it has mixed pattern of senescence with regard to ethylene sensitivity and well-defined abscission processes. Its flowers have an extremely short life (approximately 16 hours from opening to wilting) and a regeneration technique has been standardized. Moreover, a number of genes that are highly up- and down-regulated during flower opening, flower senescence and during acquisition of abscission competence have been identified. To determine the functional importance of some of these genes, we sought to use the TRV based VIGS system in M. jalapa, but despite multiple approaches, we were unable to obtain a silencing phenotype with a fragment of the gene encoding M. jalapa phytoene desaturase (PDS). In investigating possible reasons for the failure of the VIGS system in M. jalapa, we noted that tubers of this plant contain high levels of a well-studied antiviral protein named Mirabilis Antiviral Protein (MAP). This protein is present in high concentrations in all parts of the plant, and has been shown to have potent activity in preventing viral infection and inhibiting protein synthesis through its activity as a type I ribosome inactivating protein. We reasoned that since this protein inhibits normal viral replication, it may also prevent virus-induced gene silencing.
We report here successful VIGS-based gene silencing in M. jalapa, using a TRV vector containing a fragment of the MAP gene. M. jalapa plants inoculated with a TRV vector containing fragments of PDS and MAP (to silence the antiviral protein) resulted in the typical photo-bleaching phenotype in photosynthetic tissues. We demonstrated reduced transcript abundance (and the expected phenotype) when we used VIGS with the TRV/MAP vector to silence a range of genes. Silencing 3,4-dihydroxy-L-phenylalanine 5-glucosyl transferase (DOPA 5GT) reduced pigmentation in stems, leaves, and flowers. This is the first direct demonstration of the role of enzymes that have been proposed to be key steps in the synthesis of betalain pigments. Silencing endogenous antiviral proteins may open a wider range of taxa to the use of VIGS for gene function analysis.